ABSTRACT
The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.
Subject(s)
Blood Pressure/physiology , Protein Serine-Threonine Kinases/deficiency , Animals , Base Sequence , Blood Pressure/genetics , DNA, Complementary/genetics , Gene Library , Genetic Techniques , Heterozygote , Humans , Hypertension/therapy , Intracellular Signaling Peptides and Proteins , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Minor Histocompatibility Antigens , Molecular Sequence Data , Mutagenesis, Insertional/methods , Phenotype , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , Sequence Tagged Sites , WNK Lysine-Deficient Protein Kinase 1ABSTRACT
Maltose metabolism in Lactococcus lactis involves the conversion of beta-glucose 1-phosphate to glucose 6-phosphate, a reaction which is reversibly catalysed by a maltose-inducible and glucose-repressible beta-phosphoglucomutase (beta-PGM). The gene encoding beta-PGM (pgmB) was cloned from a genomic library of L. lactis using antibodies. The nucleotide sequence of a 5695 bp fragment was determined and six ORFs, including the pgmB gene, were found. The gene expressed a polypeptide with a calculated molecular mass of 24210 Da, which is in agreement with the molecular mass of the purified beta-PGM (25 kDa). A short sequence at the N-terminus was found to be similar to known metal-binding domains. The expression of beta-PGM in L lactis was found to be induced also by trehalose and sucrose, and repressed by lactose in the growth medium. This indicates that beta-PGM does not serve solely to degrade maltose, but that it is also involved in the metabolism of other carbohydrates. The specific activity of beta-PGM during fermentation was dependent on the maltose concentration in the medium. The maximum specific activity of beta-PGM increased by a factor of 4.6, and the specific growth rate by a factor of 7, when the maltose concentration was raised from 0.8 to 11.0 g l-1. Furthermore, a higher amount of lactate produced relative to formate, acetate and ethanol was observed when the initial maltose concentration in the medium was increased. The specific activity of alpha-PGM responded similarly to beta-PGM, but the magnitude of the response was lower. Preferential sugar utilization and alpha- and beta-PGM suppression was observed when L. lactis was grown on the substrate combinations glucose and maltose, or lactose and maltose; maltose was the least-preferred sugar. In contrast, galactose and maltose were utilized concurrently and both PGM activities were high throughout the fermentation.
